Basic fructan info

This page will allways remain under construction

Introduction

Fixed carbon can be used for structural carbohydrate synthesis like cellulose, xyloglucan, pectin or for the synthesis of non-structural storage carbohydrates like sucrose, starch or fructan. From the three types of non-structural carbohydrates starch is the most common.
Starch consists of two polymer types; amylose and amylopectin. Both types contain glucose residues that are mainly linked to each other via alpha(1->4) bondages. Amylose is a linear molecule with only a few braches, while amylopectin is a molecule with a lot of alpha(1->6) branches. The linear glucose chains in amylopectin are synthesized by soluble starch synthase, whereas amylose is synthesized by granule-bound starch synthase. This latter enzyme will be discussed a bit further later on.
Fructan is the third non-structural carbohydrate that can be found in plants. These carbohydrates are the dominant carbohydrate reserve in several families of the plant kingdom and will be discussed in more detail below.

Fructans are polymers of fructose. They were first described by Rose, a german scientist, who found “a peculiar substance from plan origin in a boiling water extract from Inula helenium in 1804 (Rose, 1804). This substance was named inulin by Thompson in 1818 (Thompson, 1818). These events are considered to be the start of fructan research, which almost spans two centuries now. During these two centuries fructan research has expanded from basic to a more applied science, especially in the last decade.

Fructan types

Fructans have a general structure of a glucose linked to multiple fructose units. In plants up to 200 fructose units can be linked in a single fructan-molecule. Bacterial fructans can consist of up to 100.000 fructose units. There are several types of fructans present in nature. These types are distinguished on the basis of the glycosidic linkages by which the fructose residues are linked to each other. They can broadly be devided into 3 groups. The first group are the inulins, which are linear fructans, where the fructose units are (mostly) linked via a beta(2->1) bond (Carbohydrate chemistry) (Pontis and del Campillo, 1985). All fructans found in the dicotyledons, as well as some monocotyledons are of this type. The picture below shows 1-kestose (3D model). This is the shortest fructan of the inulin type. In larger fructans the coupling of fructose units is similar to the second fructose link to the first in the picture.
The second group are the levans, which are also linear fructans, but where the fructose units are (mostly) linked via a beta(2->6) bond (Suzuki and Pollock, 1986). This type of fructan is found in a large part of the monocotyledons and in almost all bacterial fructans. The picture below shows 6-kestose (3D model). This is the shortest fructan of the levan type.
The third group are the fructans of the mixed type, which are also referred to as the graminan type (Carpita et al., 1989) (Lewis, 1993). These fructans have both beta(2->1) and beta(2->6) linkage bonds between the fructose units, and thus contain branches. These fructans are found in grasses for example. The picture below shows 6,6&1 kestopentaose (3D model) (Nomenclature). This is one of the simplest fructans of this type.
In plants fructans are naturally present in the vacuole (Darwen and John, 1989), in contradiction to starch which is stored in plastids.

Fructans in plants

Fructans are polymers of fructose. They are naturally produced by 15% of the flowering plant species (Hendry and Wallace, 1993). From an evolutionary point of view fructans tend to be present within some of the most advanced families. Examples of families which contain fructans are; Liliales, Poales, Astrales, Campanulales, Palemoniaceae, Ericales and Dipsacales. Also important crops like wheat, barley, union and chicory contain fructans. It has been estimated that as much as one third of the total vegetation on earth consists of plants that contain fructans (Hendry and Wallace, 1993). A large part of the fructan producing species is present in regions with seasonal drought or cold. It has been proposed that the function of fructan in plants could be the protection against drought as an osmoprotectant and cold for example as a cryoprotectant.

Fructan-metabolism in plants

The general enzymatic model for fructansynthesis in plants is the SST/FFT model (Edelman and Jefford, 1968). The two key enzymes in this model are SST, which stands for sucrose-sucrose-fructosyltransferase, and FFT, which stands for fructan-fructan-fructosyltransferase (Smeekens, 1991). These enzymes are usually found in the vacuole. SST catalyses the reaction that that makes a trisaccharide and a glucose from two sucrose molecules.

GF + GF -> GFF + G

Recently it has been found that SST can also make DP4 (degree of polymerization) and DP5 (Koops et al., JAAR). FFT, the second enzyme, is the molecule that builds larger fructans. It uses the trisaccharide that is formed by SST for further chain elongation of the fructanpolymer by which a sucrose molecule is released. SST and FFT thus have some overlapping activities.

G(F)n + G(F)m -> G(F)n-1 + G(F)m+1

FFT can use any fructosepolymer, including sucrose as an acceptor. Through the chain elongation by FFT a degree of polymerization (DP) higher than three is achieved. Different FFT enzymes have been identified. The FFT enzymes are probably also responsible for the structural diversity of fructans that are found in nature, because these enzymes are responsible for fructans with more that two fructose units. Other enzymes that are involved in fructan metabolism are fructanhydrolyses (FH), and Invertases. Fructanhydrolase, of which two enzymes are already isolated, is a beta-fructofuranosidase and can uncouple fructose units from fructans with sucrose as endproduct (Pollock, 1986). Invertase which is active in the vacuole, cleaves one sucrose molecule in a glucose and a fructose molecule. It's also capable of cleaving fructose molecules from smaller fructans. This fructandehydrolysing activity decreases with a higher degree of polymerization of the fructan.

Function of fructans

Plants with fructans are especially abundant in areas with seasonbound or sporadic rainfall in an annual cycle (Hendry and Wallace, 1993). It could be that the evolutionary meaning of fructanaccumulation is a adaptation to drought. That is, fructans are soluble in water in contrast with starch, and is therefor osmotically active. By changing the DP of the fructans in the vacuole, the plant can quickly change the osmotic potential of it's cells without having to alter the total amount of carbohydrates. It has been shown that transgenic tobacco plants transformed with CPY-LS (a construct containing the bacterial gene levansucrase to produce fructan), put under polyethyleneglycol-mediated drought stress, had an increased growth rate, fresh weight and drought weight over wildtype plants (Pilon-Smits et al., 1995) There are also indications that this osmotic activity of fructans is involved with cold-stress. Incontrovertible evidence for another possible function than that of storage sugar of fructan is still not found.

Fructans in bacteria

Some species from a number of bacterial genera have been reported to produce fructans. In the following genera fructan producing species are found: Tolypothrix, Pseudomonas, Xanthomonas, Azotobacter, Erwinia, Streptocuccus, Bacillus, Actinomyces, Rothia and Arthrobacter. (Hendry and Wallace, 1993) Bacterial fructans are often much larger than fructans produced by plants. They can achieve a degree of polymerization of 100.000 or more fructose units (Lieberman et al., 1976). The structure of most of the fructans produced by bacteria is of the levan type, consisting of beta(2->6) linkages with an occacional beta(2->1) branch. In contradiction to the plant genes of enzymes involved in fructan metabolism, bacterial genes for the enzymes are cloned and their sequence defined. In bacteria only one enzyme is responsible for the synthesis of trisaccharides and the further elongation of fructans, in contradiction to the SST/FFT model in plants. Examples of two enzymes that have been studied extensively, are levansucrase (LS) and fructosyltransferase (FTF). Levansucrase which produces levans was isolated from Bacillus subtilus (Dedonder, 1966) and fructosyltransferase, which produces inulins was isolated from f (Carlsson, 1970). The levansucrase enzyme catalyzes the following reaction:

GF +G(F)n -> G + G(F)n+1
sucrose + levan(n) -> glucose + levan(n+1)

Levansucrase also has invertase (hydrolase) activity, in which it catalyzes the following reaction:

GF + H2O -> G + F
sucrose + water -> glucose + fructose

Site directed mutagenesis experiments have showed that the hydrolysing and polymerizing activities can be modulated seperately (Chambert and Petit-Glatron, 1991). It has also been shown that the hydrolizing activity of levansucrase can be diminished in the presence of high concentrations of non-aqueous solvents (Chambert and Petit-Glatron, 1989). In Bacillus subtilis the enzyme is synthesized in the cytoplasm and excreted into the extracellular space, where it catalyses the reactions described above. A two step process has been proposed for the maturation of levansucrase (Petit-Glatron et al., 1987). The maturation involves the removal of a signalpeptide and the proper folding of the enzyme catalyzed by iron ions (Petit-Glatron et al., 1987). The molecular weight of the mature levansucrase is 50 kDa (Steinmetz et al., 1985), the Km for sucrose is 27 mM and the enzyme is active between pH 3,0 and pH 8,5 with an optimum at pH 6,0 (Dedonder, 1966).

Applications

Fructans can be applied in the foodindustry and in other industries, for example as washsoftener or biologically degradable plastics. You can already buy dairy-products with inulins. Fructans can also be used as low calory sweeteners. Humans are not able to degrade fructans, but they do have a sweet taste. Also the fructose which is produced by the degradation of fructan can be used as low calory sweetener. Fructose is 1.6 times as sweet than glucose. Fructans can also be used as substrates in the production of ethanol or glycerol for example. As source for the production of fructans, plants or bacteria can be used. At this point inulin production from cichory is the only profitable, and applied application. Disadvantages of existing fructanflora are the low degree of polymerization and the degradation in storage. On top of that chichory has a low harvest index (harvestable weight per plant) compared to the more widespread agricultural crops like patato of tabacco. It would be desirable to introduce fructanaccumulation in agricultural crops. By introduction of bacterial genes accumulation of fructans with a high DP, that were not degraded by the plants, is achieved in tabacco and in patato. This is what the research in Utrecht is about. Next to use for industrial production of fructans, there might be interesting applications possible for these transgenic plants in agriculture. Fructan accumulation can be used to achieve a higher dryweight of harvestable parts of plants. Fructan accumulation might also give drought tolerance, or cold resistance to crops. From a scientific point of view transgenic fructanaccumulating plants are interesting for research in the function of fructans in plants and the effect on carbohydrate metabolism due to the introduction of fructans.

Last updated on 20/12/1996

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